Hepatitis C virus (HCV) is a positive-strand RNA virus that infects more than 170 million people worldwide. The infection mainly affects the liver and leads to both acute and chronic liver diseases including cirrhosis and hepatocellular carcinoma (HCC). HCV replication has proved to be a good model system for studies of virus-host cell interactions, as the virus often establishes a chronic infection that typifies the intricate relationship between a pathogen and its host.
Research on HCV infection in vitro has been hampered by lack of an efficient cell culture-based production and infection system. The very recent reports of production of infectious HCV particles in tissue culture will hopefully break through this barrier and usher in a new era of HCV research (13, 24, 30). Studies of HCV RNA replication, however, received a great boost with the development of the HCV subgenomic replicon system a number of years ago (1, 14). Many important questions about the RNA replication of the virus were answered through use of the replicon cells. Adaptive mutations that permit efficient RNA replication in cultured hepatoma cells as well as nonhepatic cell lines have been defined (1, 11, 31); critical components of replication have been mapped, and the data greatly complemented the limited in vivo data available previously (4, 10, 29). A novel mechanism by which HCV suppresses the innate antiviral responses to establish persistent replication has been uncovered (3). The replicon system is also being used extensively to evaluate antiviral agents such as small-molecular drugs (8) as well as small interfering RNAs (21). The validity of the replicon system for this purpose has been highlighted by the in vivo efficacy in humans of a small-molecule compound that was tested solely in the replicon without any animal efficacy study (12). Even though the future of the compound is uncertain because of in vivo toxicity issues, the excellent correlation between the replicon inhibition in vitro and the in vivo antiviral effect provided good proof of principle for both the class of the drug and the screening assay.
Various derivatives of the HCV replicon that harbor different reporter genes have been constructed to facilitate the measurement of replication by means of surrogate markers (11, 18, 28). Recently, a replicon cell line has been developed in which the green fluorescent protein (GFP) gene is inserted into the coding region of the NS5A gene without abolishing the ability of the RNA to replicate in Huh-7 cells (16). This line allowed direct visualization of fusion protein, NS5A-GFP, with fluorescence microscopy for study of the dynamics of the HCV replication complex in living cells, as NS5A is one of the nonstructural proteins believed to be in the replication complex. Co-localization of NS5A and newly synthesized viral RNA was observed.
A tight coupling between HCV replication and the physiological state of the host liver cells has been observed. In particular, a highly reproducible and reversible inhibitory effect of cell confluency on HCV replication was reported: high levels of HCV RNA and protein that can be detected in actively growing cells decrease dramatically when the replicon cells reach confluency. When the confluent cells are split into lower density, replication resumes, and HCV expression recovers (20, 22).